Patent Application
20110134421
The present invention relates to apparatus and a method for detection of Raman signals in electromagnetic radiation. It finds particular application in the detection of a Raman signal in the presence of background which could otherwise mask it.
Raman spectroscopy is used in the study of vibrations of atoms or molecules in a sample. Light can be considered to be made up of particles called photons. When a photon is incident on matter, one of a number of different events can occur. The photon can pass through unchanged, it can be absorbed, or it can be scattered. In the case of scattering, the photon's direction of travel is changed by the scattering event. Usually, the photon's energy is unchanged—this is called elastic scattering. Sometimes the photon's energy is changed—this is called inelastic scattering, or Raman scattering. The magnitude of the change in energy is exactly equal to the energy of a vibration of the matter. Since the ‘allowed’ energies (frequencies) of vibration usually form a well defined set of discrete values, the spectrum of the inelastic scattered light also usually exhibits a set of one or more well defined values (in practice bands known as Raman bands) and these are usually characteristic of the matter concerned.
A Raman spectrometer measures the spectrum of the scattered light. This is usually plotted as intensity (photon counts) vs. wavenumber shift (measured in cm−1, which is proportional to change in photon energy from the incident to the scattered photon). The wavelength of the incident photon can, in principle, be chosen to be anything. In practice, visible, or near-visible wavelengths are usually chosen. The shorter the wavelength (the higher the photon energy), the greater the Raman signal; the un-enhanced Raman signal (measured in photon number) is approximately proportional to the 3rd power of the excitation photon energy. Typical un-enhanced Raman efficiencies are 10−8 to 10−12 (measured as Raman photons out per excitation photons in).
If the incident photon is absorbed by the matter, or material, then one of the events that may follow is for the material to emit another photon (usually of lower energy). This is sometimes called luminescence. Some of these emitted photons may be indistinguishable from Raman scattered photons, although there is often a small time difference between the absorption and emission processes.
Many ‘real-world’ samples contain contamination from other materials. Often these other materials, or even the sample of interest, will exhibit a degree of luminescence that may overwhelm the Raman signal.
Raman spectrometers typically consist of a source of monochromatic light (usually a laser), some method for delivering this light to a sample, some method for collecting the scattered/emitted light from the sample, a method to filter out the elastically scattered component, and a method for analysing the remaining light to determine the light intensity as a function of its photon energy (wavelength, or colour).
Advances in technology during the late 1980s and early 1990s enabled efficient Raman spectrometers to be made. These advances included very efficient charge coupled device (CCD) cameras and very efficient holographic notch filters. More recently, developments have included dielectric filters, lasers to enable Raman spectroscopy to be carried out over a wider range of excitation wavelengths, and small improvements to the efficiency of diffraction gratings and CCDs. Also, the optics for coupling the light to/from the sample have evolved and now optical fibres are commonly, but not exclusively, used for this purpose.
One of the main limitations of the use of Raman spectroscopy in real-world applications is interference from luminescence. Luminescence (be it fluorescence, phosphorescence, or some other mechanism) is usually much more efficient than Raman scattering. However, efficient luminescence occurs when the incident photon energy lies within (or near to) an electronic absorption band of the sample. These absorption bands are fixed in wavelength for a given material and environment. Hence, when dealing with a luminescent sample, it is common practice to choose an excitation wavelength that is inefficient for luminescence, but still provides reasonable Raman scattering.
By choosing an excitation wavelength that is towards the short wavelength (high photon energy) end of the spectrum, one can avoid (some of the) luminescence as this may occur at longer wavelengths compared to resultant Raman scattering. The Raman scattering will be more efficient, and may also be enhanced by resonant processes. However, the optics are generally more challenging to make (hence more expensive) and the high excitation photon energy can damage the sample. Choosing an excitation wavelength that is towards the long wavelength (lower photon energy) end of the spectrum, luminescence may again be weak as the incident photons do not have sufficient energy to excite the electronic states, but the Raman scattering will also be weaker. It is therefore, from the point-of-view of Raman scattering, often preferable to use an excitation wavelength somewhere away from the extremes of the near-visible spectrum, but this often results in a significant amount of luminescence.
Raman bands are typically a few cm−1 wide (say 2 cm−1 up to 10's cm−1). Luminescence emissions are usually many tens of nanometres, up to ˜100 nm wide. With typical visible excitation wavelengths, 1 nm≈20 to 40 cm−1. Hence, luminescence emission bands are roughly two orders of magnitude or so broader than Raman bands.
Within a typical Raman spectrum, any luminescence appears as a smooth background level, perhaps with some curvature. Background subtraction techniques are known which are used to remove this background and leave the Raman signal as a series of bands above a (near) zero baseline. These known techniques subtract a smooth curve; they do not subtract the noise that is associated with the background. The Raman signal has to compete with this noise level. A large background level will have a large noise level which may still overwhelm a weak Raman signal, perhaps making the Raman bands undetectable.
According to a first aspect of embodiments of the present invention, there is provided apparatus for detecting one or more Raman bands in radiation emitted by a sample in response to illumination which oscillates in wavelength, the emitted radiation being detected as a set of discrete detection signals over a wavelength range, the apparatus comprising a signal processor arranged to:
It has been recognised that it is possible to enhance a spectral representation of Raman bands preferentially over other components such as noise by a relatively simple processing of a set of derived mean values over time as produced in step “b” above. The detection signals are spectrally related because they're produced as discrete measurements across a wavelength range of the emitted radiation. The time-dependent intensity signals and the derived mean values can also thus be spectrally related. A series of the derived mean values usable to create a spectral representation of the emitted radiation has distinctive characteristics where a Raman band lies and these distinctive characteristics allow relatively simple processing of the derived mean values to produce enhancement of the appearance of the Raman band in that spectral representation.
One way of selecting and combining the at least two derived mean values is to select values having a specified polarity, such as negative values, and shift them to a different spectral location in the spectral representation, adding their magnitudes to those of any positive values relevant to that spectral location. Generally, if the spectral shift brings the selected values to a position in the spectral representation where there are values whose magnitudes can be added, there will be enhancement of the appearance of the Raman band. This is because the overall effect of using the derived mean values of step “b” is that noise and other background will tend to be relatively close to zero compared to the distinctive characteristics where the Raman band lies and thus shifting and adding the magnitudes of the derived mean values will only tend to have a significant enhancing effect for the Raman band values.
Another way of selecting and combining the at least two derived mean values is to integrate a consecutive series of derived mean values. In this case, the selection of mean values may simply be done by selecting each next derived mean value in the series and adding it to an existing total for the previous mean values.
In practice, embodiments of the present invention function particularly effectively where a Raman band to be detected has a width which is comparable to the wavelength range of the oscillation of the illumination. An embodiment of the invention may thus further comprise an illumination source for illuminating the sample, which source is controllable to oscillate in wavelength over a wavelength range of the same order as the width of at least one Raman band to be detected. An effective wavelength range in any particular experimental arrangement can be discovered by trial and error. However, in an example, “of the same order” might mean here not more than five times the width, or more preferably within perhaps 50% or less. If the wavelength range is more than ten times the width of a Raman band to be detected, then the band is likely to be undetectable.
In order to detect the Raman band(s), the emitted radiation needs to be detected over a wavelength range which is offset from the wavelength range of the illuminating radiation. An embodiment of the invention for use with a source controllable as above may thus further comprise a wavelength-sensitive detection arrangement for detecting the intensity of electromagnetic radiation emanating from the sample at a plurality of detection wavelengths or wavelength ranges, offset from the illumination wavelength range, to give said set of discrete detection signals.
Putting the above elements together, an embodiment of the present invention might comprise apparatus for performing Raman spectroscopy by generating data values for respective spectral elements of a spectral representation of one or more Raman bands, the apparatus comprising:
Embodiments of the invention allow a previously ‘undetectable’ Raman signal to be detected and measured, despite being overwhelmed by background noise, or equivalently a signal that appears weak compared to the background noise to improve its signal-to-noise ratio.
Step “a”, which is extracting a set of time-dependent intensity signals from respective detection signals, might be done by subtracting the mean value of the detection signal over time to create a baseline-corrected time-dependent signal that varies around zero. This removes the contribution to the detection signal of the general background level which is not time-dependent.
Then in step “b”, which is determining components that have a temporal correspondence to illumination of the sample, might comprise multiplying the baseline-corrected signal by a reference signal which comprises at least one frequency which is related to a frequency of the wavelength oscillation of the illumination. The required component of the signal may then be found by determining the mean value (over time) of this product of the baseline-corrected signal and the reference signal. For example the reference signal might comprise a fundamental frequency of the wavelength oscillation and/or a harmonic thereof. This effectively removes much of the noise associated with the background level.
Step “c” recognises that, as long as the range of the wavelength oscillation of the illumination is comparable to the width of the Raman bands to be detected, the output from step “b” (the mean value over time of the product of the baseline-corrected signal and the reference signal) can create a distinctive derivative of the Raman band spectrum which can then be summed or integrated in a way that exaggerates the Raman bands in a spectral representation of the data, making them more detectable. For example, if the reference signal comprises a fundamental frequency of the wavelength oscillation of the illumination, the distinctive derivative appears for each Raman band as a “switchback curve” with a positive peak followed by a negative trough. If the values in the switchback curve are numerically integrated, this produces an exaggerated peak centred on the middle of a Raman band. Alternatively, if the moduli of the values in the positive peak and the negative trough are shifted towards each other to overlap, again this gives an accurately positioned and exaggerated peak representing the Raman band. This can be done by shifting the modulus of each value by the same fixed offset value, the direction of offset being determined by whether the value comes from the positive peak or the negative trough.
If the reference signal comprises a first harmonic frequency of the wavelength oscillation of the illumination, the distinctive derivative appears for each Raman band as a central peak with lateral troughs. These values can still be used in detecting a Raman band by taking the magnitude of the values in the central peak and summing the moduli of the values from the lateral troughs but shifted by a fixed offset value towards each other to overlap the central peak. For example, the fixed offset value might bring the lateral troughs to sit at the centre of the central peak. In this case, it is necessary to distinguish which lateral trough is which so that the moduli of the values from each trough can be shifted in the correct direction. This can be done by referring to the “switchback curve” mentioned above, relating to the fundamental frequency. The positive and negative parts of the switchback curve will map onto different respective lateral troughs and can thus be used to distinguish them.
The fixed offset values mentioned above in relation to step “c” are preferably, in both cases, close to or equivalent to the amplitude of wavelength oscillation of the illumination. As long as the peak-to-peak range of that wavelength oscillation is approximately equal to the width of the relevant Raman band, this preserves a degree of accuracy in terms of width of the Raman band in a representation of it based on step “c”. Also, if the illumination oscillates across a wavelength range at least approximately equal to the spectral range of a Raman band to be represented, it generally means the “distinctive derivative” of the Raman band is most pronounced.
The wavelength variation of the illumination and/or the reference signal can be represented by, for example, a smooth sine wave meaning the reference signal traverses to and fro across the wavelengths in a wavelength range in a continuous manner; or it can be represented by a discrete sine wave, meaning the reference signal traverses to and fro across the wavelengths in a wavelength range repeatedly showing the wavelengths towards the ends of the range plus at least one intervening wavelength; or it can be represented by a square wave in which the reference signal switches between the two wavelengths at the ends of the range.
In embodiments of the invention according to its first aspect, the wavelength sensitive detection arrangement preferably receives electromagnetic radiation across a spectral range greater than the width of a Raman band. This allows it to produce intensity signals across the Raman band while the band itself moves across the wavelength sensitive detection arrangement. These intensity signals still contribute by means of step “c”, combining the magnitudes of the derived mean values for two or more detection signals, to the representation of a Raman band.
The reference signal can be supplied, in use, to the illumination source so as to generate the wavelength oscillation of the illumination. This ensures a frequency match when it comes to analysing the detection signals. However, it is not essential to use the same signal.
A spectral representation of a Raman band can include data derived from both the fundamental reference frequency and the first harmonic of the reference frequency. It is noted that higher harmonics may also be used to refine the resultant spectrum further.
In embodiments of the invention, it is recognised that oscillating the excitation radiation in wavelength creates an output from the sample in which the Raman bands are oscillated to and fro spatially over an array of detectors. The spatial scanning of these Raman bands necessarily has frequency components related to the oscillated excitation radiation, making the bands detectable. By processing the time-resolved responses of individual detectors against their respective wavelengths, one can create a direct representation of a Raman band in a spectral display.
As mentioned above, it is preferable that the peak-to-peak wavelength range encompassed by the oscillation of the illumination is of the same order as the width of a Raman band it is intended to detect as this enhances the signal to noise ratio of the Raman signal. If the wavelength range is too great or too small, for example by a factor of ten with regard to the width of the Raman band, the Raman signal may become undetectable. It may be preferable to use a more complicated wavelength variation function, for example a superposition of two or more oscillations each with different amplitude “a” and with different temporal frequencies. This allows the selection of a representation of a Raman band which has been achieved with the best match between the wavelength range and the width of the band. It also deals with the potential difference in width of the Raman bands for a single sample, allowing one analysis operation to give efficient detection of differently sized bands.
According to a second aspect of the present invention, there is provided a method of detecting one or more Raman bands in radiation emitted by a sample in response to illumination which oscillates in wavelength, the emitted radiation being detected as a set of discrete detection signals over a wavelength range, the method comprising:
Embodiments of the invention in its second aspect may additionally carry out any or all of the steps described above in relation to the invention in its first aspect, such as illuminating the sample with radiation which oscillates in wavelength over a wavelength range of the same order as the width of at least one Raman band to be detected. In another example, an embodiment of the invention in its second aspect may comprise detecting the intensity of electromagnetic radiation emitted by the illuminated sample at a plurality of detection wavelengths or wavelength ranges, each detection wavelength or wavelength range being outside a wavelength range over which the sample is illuminated, to give said set of discrete detection signals.
Any feature described in relation to one aspect or to any one embodiment of the invention may be applied in relation to one or more other aspects or embodiments of the invention if appropriate.
Raman detection apparatus according to an embodiment of the present invention will now be described, by way of example only, with reference to the accompanying figures in which:
It should be noted that the figures are not drawn to scale.
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The digitised data output from the ADC 145 can then be processed by a detection signal processor 155 embodied in software installed on a computing platform 130.
It might be noted that the amplifier 140 and ADC 145 are usually integrated into a CCD (“charge-coupled device”) camera, and are ‘transparent’ to the end user. The basic requirement is just for a (preferably linear) array detector, which is conveniently provided by a CCD type detector but could be anything that acts as an array of detector elements.
In outline, embodiments of the invention use an illumination source of electromagnetic radiation 105 that can be oscillated in wavelength by a few cm−1 (i.e. of the order of the width of a Raman band). The measured spectrum received at the detectors 120 will consist of a nearly static background signal, its associated noise, and a superimposed Raman signal which oscillates along the spectral axis in synchronisation with the oscillation of the illumination source 105. By measuring the resultant signal that is oscillating in synchronisation with the light source, a weak Raman signal can be extracted from a large, noisy background since the background should not have a particular frequency response—that is, it will be white noise. Embodiments of the invention use methods to extract the components of the signals at the detectors that vary in synchronisation with the illumination source 105, and then to assemble these by summing or integration to reconstruct the Raman signals even where previously the Raman signal was undetectable above background noise.
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Each laser 105 has a narrow spectral range, that is, significantly less than the width of a Raman band to be detected and perhaps for example sub 1 cm−1. Some laser technologies emit much narrower lines than this and could equally well be used.
More than two lasers 105 may be chosen, each emitting radiation at different wavelengths. The radiation incident on the sample 100 can be chopped between/among these different lasers 105.
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The wavelength range of each detection channel 125 needs to be small enough to be able to give a sufficiently high sampling rate across detected Raman bands for the Raman bands to be adequately re-constructed. For example, a reasonable number of detector elements 120 would give several, say five to twenty, detector elements 120 covering a typical Raman band. A known type of detection arrangement that would be suitable is a dispersive spectrometer. Physically, most dispersive spectrometers spread incoming light into a line, in which one end of the line corresponds to short wavelengths and the other end of the line to long wavelengths. Detector elements 120 also have a finite physical size (active area). The two sides of each detector element 120 correspond to slightly different wavelengths, with a continuous spread of wavelengths between. This determines the pixel resolution of the spectrometer. If one spread the light into a wider line, or used a detector with smaller elements 120, then the pixel resolution would improve. In practice, one also typically has an entrance slit on the spectrometer. This slit acts as a finite aperture in the dispersive direction. If one has a narrow slit, one can get the spectrometer to be limited by the pixel resolution, but one will not get much light through the slit. So one broadens the slit to let more light in, but the image of the slit on the detector elements 120 can be a size more than one element. The spectral resolution is now limited by the slit width.
One could use a spectrometer in which there were more than twenty detector elements 120 covering a single Raman band but to get most of the entire Raman spectrum (which usually consists of many separate bands) one may then need a much longer and possibly impractical detector.
Regarding instrumentation, there are many companies that manufacture Raman spectrometers. The usual requirements for these instruments are a narrow line-width laser (typically less than 1 cm−1), an optical filter to remove most of the Rayleigh scattered light, and an optical spectrograph with a multiple-element, low noise, high quantum efficiency detector. These components will not be described here as they are well known in the industry and in the literature. The particular features that are needed for an embodiment of the present invention are as follows:
Many different components are available to provide these features that can have a wide range of specifications. As an example, the light source can be achieved using an external cavity tuneable diode laser. One such family of lasers is manufactured by Sacher Lasertechnik. For example, the Littman/Metcalf Lion TEC 500 or the Littrow Lynx TEC120 laser are suitable. Alternatively, one can use two lasers and simply chop between them. Any pair of Raman compatible lasers can be used provided their wavelengths are spectrally separated by an appropriate amount.
For the detector, many manufactures make CCD devices that are suitable for Raman spectroscopy, and that can read out spectroscopic lines at appropriate speeds. One such camera is a Princeton Instruments Pixis 2K, which can read spectra at a rate of up to 90 spectra per second. Again, there are many other appropriate cameras in this range, and made by other manufacturers.
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Thus the responses of the five detectors A . . . E have a definite phase and harmonic frequency relationship with each other and this relationship can be exploited to enhance significantly the ability of the system to discriminate a small Raman signal from background noise.
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In practice, many more detectors would be used. In the following examples of Raman signal detection according to embodiments of the invention, intensity data that would be produced by a full array of detectors 120 is processed to create representations of Raman bands, using different reference signals and forms of oscillation of the excitation radiation.
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Instead of referring to the fundamental of the reference signal product to determine the + or − direction of the spectral bin a value will be added to, an alternative approach would be to set a flag in a memory location when a significant series of negative values had been assigned to spectral bins. A following series of negative values would then be assigned to spectral bins in the opposite spectral “direction”. However, this approach could be less dependable in processing noisy data.
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Step 7 in the case of the fundamental frequency has the effect of moving the positive and negative peaks of the switchback curve 1000 inwards, in a spectral direction for each of them that crosses the midpoint of the Raman band when the excitation source passes through the midpoint of its wavelength oscillation. This is indicated by the small arrows shown in FIG. 10. By using the moduli of the “DC” values of step 5, the negative peak is effectively inverted and added to the positive peak to obtain a narrowed resultant peak 1005 labelled as the “F” curve on
Step 8 in the case of the first harmonic has the effect of leaving the positive peak of the three-part curve 1010 where it is but bringing in the two negative portions, again as indicated by the small arrows shown in
Adding the “F” curve and the “2F” curve gives a more intense and therefore more detectable curve 1025 which is a good approximation to the Raman band of interest.
It might be noted that in practice, because data obtained in Raman spectroscopy will be stochastic, there may be spectral data bins within the Raman band that do not have any values, or only one value, in them.
Thus there is a clear enhancement in the intensities of the resultant signals from the fundamental frequency analysis when compared to the conventional method; each of these simulations used equivalent parameters (signal level, background level, integration time, noise).
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The situation with square wave excitation (for instance where the output of a laser 105 oscillates between two discrete excitation wavelengths) is very similar to that with sinusoidal oscillation of the excitation radiation and the methodology is almost the same as described above. However, in this case, only the fundamental signal is used and not the first harmonic, and a square wave is used as the reference signal instead of the sinusoidal wave. Hence the protocol for analysing the data thus obtained is the same as for steps 1 to 7 described for the sine wave reference signal.
The ‘encoded’ data from step 6 of the above protocols can be treated in different ways to provide a representation of the Raman spectrum. A second method is for step 7 to be simply to integrate this data numerically. This results in an approximation to the original Raman spectrum, wherein any narrow peaks are effectively broadened to the amplitude of the oscillation of the excitation wavelengths, and where the baseline exhibits a pseudo random walk.
In more detail, the starting point for the numerical integration of Method 2 in the case of a single Raman band would be the “switchback” curve 1000 of
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It will be understood that the method could be applied in either “direction” across the switchback curve 1000 but if it is applied to the negative section first, the result will still enhance the representation of a Raman band but as a negative trough rather than a positive peak.
The numerical integration method is carried out across the whole spectral range, not just the portions which show switchback curve character. However, only the portions with the switchback character build identifiable peaks and it is these which show the presence of Raman bands. Between the peaks, the random nature of noise tends to smooth out in the integrated curve.
The resultant spectrum does though have a ‘random walk’ baseline because the mean value in step 5 above will not be the same for all the spectral data points due to random noise and an error factor is introduced which can accumulate as the integration progresses across the spectrum. Nevertheless, the Raman bands will appear with a significantly greater signal to noise ratio than without carrying out such a protocol.
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In general, Raman bands that are intrinsically narrow will be effectively broadened by the amplitude of the oscillation of the excitation wavelength in embodiments of the invention. This is inevitable as the representation of each Raman band is always, as described above, made in relation to spectral elements which are at least spread across the peak-to-peak wavelength range of the illumination. Using numerical integration (Method 2), the Raman bands will each be spread beyond that wavelength range by half the width of the Raman band at either end of the wavelength oscillation.
The best results are achieved when the peak-to-peak wavelength range of the oscillating illumination is approximately equal to the width of the Raman band. Embodiments of the invention depend on seeing a distinctive change in the mean value of intensity measurements over time for the spectral channels 125. If the peak-to-peak wavelength range of the oscillating illumination is mismatched to the width of a Raman band, the size of the intensity changes over time will be reduced. Either the intensity will tend to stay high (Raman band broad relative to the wavelength oscillation) or it will for a larger proportion of time stay low (Raman band narrow relative to the wavelength oscillation). The biggest change in intensity, making the Raman band most detectable, is when the wavelength oscillation produces a movement in the Raman band across the detectors 120 which is of the order of its own width.
If for example an embodiment of the invention is being used to detect a Raman band of unknown width, or if it is known that there may be Raman bands present that have significantly different widths, then it may be preferable to use illumination radiation which has a relatively complex variation in wavelength. It is straightforward to envisage encoding a wavelength amplitude oscillation in the illumination radiation to incorporate several amplitudes and to use matching algorithms to extract the multiple bandwidth spectra from this mixed driver. This would make Method 1, the offset method, applicable to a broader range of Raman bandwidths. For example, one can oscillate the wavelength using a superposition function such as sine frequency P plus sine frequency Q, each with different amplitudes, then look at the resultant signal at each frequency in turn to extract the Raman band whose width best matches the corresponding amplitude. In the case of a square wave modulation of the illumination, one might incorporate a third and perhaps further wavelengths in order to incorporate oscillations of different amplitudes. Such arrangements may be thought of as analogous to amplitude modulated radio signals.
| Number | Date | Country | Kind |
|---|---|---|---|
| 0810761.7 | Jun 2008 | GB | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/GB2009/050616 | 6/3/2009 | WO | 00 | 2/18/2011 |
